The present invention relates to a polymyxin lipopolysaccharide antigen that creates antibodies that detect endotoxin and the method of making the antigen.
The invention described herein was supported in part by Cooperative Agreement 58-48YK-5-0050 from the United States Department of Agriculture.
1. Field of the Invention
This invention relates to the production and characterization of antigens that create antibodies specific for endotoxin and more specifically, the antigen and the antibodies, and use of these specific antibodies as an immunodiagnostic reagent to detect endotoxin.
2. Description of the Prior Art
Bacterial contamination is currently one of the leading causes of fatal bacterial infection. Endotoxin is known to cause toxic effects in multiple cell types and organs. Endotoxin is responsible for the symptoms associated with byssinosis, septicemia, bacteremia, and the like. These symptoms may include fever, diarrhea, hemorrhagic shock, tissue damage, bronchospasms, damage to endothelial cells and the like. Release of endotoxin into the blood stream may cause endotoxin shock as well as other systemic effects.
Endotoxin is a component of the cell wall of Gram negative bacteria. The cell wall of Gram negative bacteria is multilayered and quite complex. Endotoxin is composed of lipid A and a polysaccharide. Lipid A is the core material. The polysaccharide is exterior of the lipid A. The polysaccharide component is unique to each type of bacterium.
Assays to detect endotoxin activity have received great attention. Among the procedures currently in use to analyze solutions for endotoxin activity are the Limulus amebocyte lysate assay (LAL) and the USP rabbit test for pyrogenicity.
The LAL assay measures endotoxin by detecting only one biological activity of endotoxin in a highly purified sample. The LAL measures the ability of lipopolysaccharide to gel or precipitate the lipote. Results from the assay do not correlate with the ability of endotoxin to cause respiratory toxicity in animals or nuetrophil chemotaxis in human beings. Difficulties encountered with the LAL assay include interference by numerous organic compounds yielding both false-positive and false-negative results, as well as an inability to correlate consistently with the USP rabbit pyrogen test.
The rabbit pyrogen test involves injecting a rabbit with a sample suspected of endotoxin toxicity. The temperature of the rabbits is measured for a period of time. If a fever is produced in the rabbit, the presence of endotoxin is indicated. A gross estimate of the amount of endotoxin can be determined by the rise in temperature of the rabbit. The USP rabbit test for pyrogenicity is expensive and the results are not always accurate.
Recent attempts to quantify the endotoxin content of biological solutions have employed immunologic assays. The relatively poor immunogenicity of lipopolysaccharide compared to protein antigens has led to use of lipopolysaccharide (LPS) complexes for immunization. Several endotoxin immunizing agents are known. See, for example, U.S. Pat. Nos. 4,057,685 and 4,185,090.
Polyclonal antibodies have been produced to wild-type LPS by immunizing rabbits intravenously with heat-killed bacteria. See Carlsson et al, "Titration of Antibodies to Salmonella O Antigens by Enzyme-Linked Immunosorbent Assay" Infection and Immunity, Volume 6 (1972) pp. 703-708; Holmgren "Studies of Methods For Quantitation of Agglutinens and Precipitins of Escherichia Coli O and K Antigen" Int. Arch. Allergy, Volume 37 (1970) pp. 480-494. Additionally, covalent linking of the disaccharide antigenic determinants to bovine serum albumin (BSA) has also resulted in high titered antisera. See Ekborg et al "Artificial Disaccharide-Protein Conjugates As Immunogens For The preparation Of Specific Anti-Salmonella O-antisera" Immunochemistry, Volume 14 (1977) pp. 153-157. However, in using these procedures, antibodies were also produced to BSA or cell membrane components.
Dufer et al "Synthese d'ADN et Synthese d'anticorps dans les cellules spleniques de la Souris apres immunisation, in vivo, par le lipopolysaccharide d'Escherichia coli, modifie par la polymyxine B" Comptes Rendus Hebdom Adai Res Des Sean Ces De L'Academie Des Sciences, SERIES D VOLUME 290 (1980), pp. 699-701 disclose an antigen using a complex of LPS and polymyxin B which induces spleen cells which are said to recognize LPS.
Methods of obtaining high titered, specific antibody have not been adequate for development of a sensitive specific serological assay for endotoxin. At present there is lacking an effective immunodiagnostic test which detects the presence and amount of endotoxin in biological materials with sensitivity.
There remains, therefore, a very real and substantial need for an antigen and a process for producing and characterizing the antigen that produces antibodies specific for endotoxin, and an antibody which will detect endotoxin from bacterial species, as well as producing these antibodies in large quantities and at a low cost for use in immunodiagnostic tests to achieve highly sensitive results.